The Elongator subcomplex Elp456 is a hexameric RecA-like ATPase

Abstract

Elongator was initially described as an RNA polymerase II-associated factor but has since been associated with a broad range of cellular activities. It has also attracted clinical attention because of its role in certain neurodegenerative diseases. Here we describe the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits each show almost identical RecA folds that form a heterohexameric ring-like structure resembling hexameric RecA-like ATPases. This structural finding is supported by different complementary in vitro and in vivo approaches, including the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification, explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of the holo-Elongator complex.

Architecture of the yeast Elongator complex

“…Therefore, we focused on the characterization of endogenous tandem‐affinity purification (TAP)‐tagged 35 Elongator, which permits the purification of all six subunits directly from yeast. We constructed yeast strains carrying endogenously TAP‐tagged versions of Elp1 and Elp6 and purified Elongator using previously established purification protocols 20. Consistent with sub‐stoichiometric cellular amounts of Elp456 in vivo 28, 36, 37, purifications of Elp1‐TAP resulted in an excess of Elp123 sub‐complex 20, whereas Elp6‐TAP purifications resulted in reduced quantities but yielded highly pure, complete, and stoichiometric Elongator complex.…”

“…We constructed yeast strains carrying endogenously TAP‐tagged versions of Elp1 and Elp6 and purified Elongator using previously established purification protocols 20. Consistent with sub‐stoichiometric cellular amounts of Elp456 in vivo 28, 36, 37, purifications of Elp1‐TAP resulted in an excess of Elp123 sub‐complex 20, whereas Elp6‐TAP purifications resulted in reduced quantities but yielded highly pure, complete, and stoichiometric Elongator complex. Large‐scale preparations yielded sufficient amounts of pure Elongator complex and Elp123 sub‐complex to analyze their overall architecture and shape by EM.…”

A coordinated codon-dependent regulation of translation by Elongator

“…This study also demonstrated the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP, supporting a role of Elongator in tRNA modification. 24 Contrary to a transcription defect that can be easily assessed by high-throughput technologies, the emerging question of Elongator targets, affected at the translation level, is far from trivial. Recently, our lab used an original proteome-wide approach based on reverse protein arrays to address this question in the fission yeast.…”

Use of a Yeast TRNase Killer Toxin to Diagnose Kti12 Motifs Required for TRNA Modification by Elongator

“…However, meanwhile a robust body of evidence indicates that its genuine role in yeast lies with tRNA modification rather than transcription elongation. Accordingly, Elongator binds tRNAs and modifies anticodon wobble uridine (U34) bases [8][9][10][11][12][13][14]. Strikingly, the methoxy-carbonyl-methyl-thio (mcm 5 s 2 ) modification at U34 in some of Elongator's tRNA substrates (including tRNA Glu ) allows anticodon cleavage by the -toxin tRNase [15].…”